The present invention relates to novel glycosaminoglycan derivatives having a neurite outgrowth promoting activity and a sialidase inhibiting activity, a process for producing them, and a pharmaceutical composition containing the glycosaminoglycan derivative as an active ingredient.
Neuronal disease is generated by the growth-inhibition of neural cells, by the induction of neural cell necrosis caused by the inability of the cells to be maintained due to reduction of sugar supply or energy metabolism inhibition in the cells as a result of ischemia, or by the inhibition of neurotransmission due to degradation of neurotransmitter. Accordingly, the treatment of this disease has been made with the aim of maintaining growth of neural cells, preventing ischemia of neural cells, or maintaining energy metabolism in the neural cells. That is, it is considered that the fundamental measures for the neuronal disease are to administer neurotrophic factors as substances having a neural cell growth activity (e.g., nerve growth factor: NGF, brain-derived neurotrophic factor: BDNF, ciliary neurotrophic factor: CNTF, fibroblast growth factor: FGF etc.) from the outside or promote synthesis of the neurotrophic factors, to prevent ischemia of neural cells and reduction of energy metabolism in neural cells caused by excess production of sorbitol formed by the activity of aldose reductase, by applying an aldose reductase inhibitor, and to maintain transmission of neural cells by preventing degradation of acetylcholine through the administration of an acetylcholine esterase. However, there are problems, such as antigenicity due to purity of the substance to be administered, side effects caused by the inhibition of enzyme activity, toxicity of the substance per se to be administered, and the like.
Also, it is known that glycosaminoglycan has the activity to accelerate neurite outgrowth promotion(J. Cell Physiol., 135: 293-300 (1988)). Furthermore, it is known that heparin and derivatives thereof, periodate oxidation reduction heparin and per-sulfated heparin, have a neurite outgrowth promoting activity and are effective in treating traumatic, ischemic and toxic neuropathy (JP-A-6-157322), but substances having a sufficient activity to inhibit these diseases for applying to medicaments have not been obtained yet.
On the other hand, it is known that sialidase (neuraminidase) inhibitors, such as GS4104 (WO 96/26933), zanamivir (4-guanidino-Neu4Ac2en: WO 91/16320, WO 94/07885), and the like, are effective as antiviral agents, particularly as influenza treating agents.
Since heparin and derivatives thereof are biological substances, they have a low antigenicity, but their use and concentration in using as medicaments are considerably limited due to their high anticoagulation activity. Generally, when heparin is administered to the living body, bleeding activity due to the high anticoagulation activity becomes a serious problem. Accordingly, a new substance having a low anticoagulation activity and a reinforced neuropathy improving effect is expected.
In order to resolve these problems, the present inventors have conducted intensive studies with the aim of obtaining a substance having a low anticoagulation activity for blood and an excellent neuropathy improving effect, and found, as a result of the efforts, that glycosaminoglycan derivatives having a specified structure have a high neurite outgrowth promoting activity, namely a neuropathy improving activity, and have accomplished the present invention by confirming that the anticoagulation activity of this substance is more sharply reduced than heparin. That is, it was found that novel glycosaminoglycan derivatives having a repeating unit structure of two saccharides of hexosamine and hexuronic acid as the backbone structure, in which the bondage between the 2- and 3-position carbon atoms of hexuronic acid as its constituting monosaccharide is partially cleaved and at least a part of the uncleaved hexuronic acid has no sulfate group on its 2-position, has a low anticoagulation activity and an excellent neurite outgrowth promoting activity. A pharmaceutical composition having an excellent effect for improving neuropathy using these properties can be provided. Additionally, based on the fact that a sialidase activity is increased and ganglioside known to have a relation to the maintenance and acceleration of a neurotrophic factor activity on the cell membrane is reduced when cell death and involution of neurite occur, the present inventors have conducted further investigations and found that the novel glycosaminoglycan derivative has a strong sialidase inhibition activity to provide a sialidase inhibitor using this inhibition activity of the glycosaminoglycan derivatives.
The first embodiment of the present invention relates to novel glycosaminoglycan derivatives having the following properties (a), (b) and (c) and comprising at least one structure represented by general formula (1) described in (d) per molecule of a backbone structure formed by a repeating unit structure of hexosamine and hexuronic acid:
(a) mol % of 2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-xcex1-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose (hereinafter also referred to as xe2x80x9cxcex94DiHS-tri(U,6,N)Sxe2x80x9d) being from 0 to 10%, mol % of 2-deoxy-2-sulfamino-4-O-(4-deoxy-xcex1-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose (hereinafter also referred to as xe2x80x9cxcex94DiHS-di(6,N)Sxe2x80x9d) being from 95 to 70%, and mol % of 2-deoxy-2-sulfamino-4-O-(4-deoxy-xcex1-L-threo-hex-4-enopyranosyluronic acid)-D-glucose (hereinafter also referred to as xe2x80x9cxcex94DiHS-NSxe2x80x9d) being from 5 to 20%, in a disaccharide composition obtained by a disaccharide analysis through a combination of degradation by a glycosaminoglycan degradation enzyme with analysis by high performance liquid chromatography,
(b) activated partial thromboplastin time (APTT) when measured by adding to standard blood plasma at a final concentration of 3 xcexcg/ml being 50 seconds or less,
(c) weight average molecular weight being from 9,000 to 13,000 Da (dalton), and
(d) general formula (1): 
(wherein R1represents H or SO3H; and R2 represents COCH3 or SO3H).
The second embodiment of the present invention relates to a process for producing a novel glycosaminoglycan derivative having the properties (a), (b) and (c) and having the specified structure defined in (d), comprising the following steps (i) and (ii) described below; and the third embodiment of the present invention relates to a pharmaceutical composition comprising the glycosaminoglycan derivative as an active ingredient:
(i) a step in which a sulfated glycosaminoglycan having a repeating unit structure of hexosamine and hexuronic acid as a backbone structure is subjected to a cleavage treatment to cleave between only the 2- and 3-position carbon atoms of at least a part of hexuronic acid having no sulfate group on the 2-position in the backbone structure, and
(ii) a step in which the product of the step (i) is subjected to a desulfation treatment by a desulfation method capable of specifically removing the 2-position sulfate group of hexuronic acid to desulfate 90% or more of sulfate groups of the total hexuronic acid having a sulfate group at the 2-position.
The present invention is described further in detail in the following.
As described above, the substance of the present invention is a novel glycosaminoglycan derivative which has the properties (a), (b) and (c) and also has the specified structure defined in (d).
The term xe2x80x9chexosaminexe2x80x9d as used herein means a monosaccharide in which the 2-position of hexose has an amino group, an acetylamino group or a sulfamino group and the 6-position hydroxyl group is optionally sulfated, the term xe2x80x9chexuronic acidxe2x80x9d means a monosaccharide in which the 6-position carbon atom of hexose forms a carboxyl group and the 2-position hydroxyl group is optionally sulfated, the term xe2x80x9cglycosaminoglycanxe2x80x9d means a polysaccharide which has a structure formed by a repeating unit of hexosamine and hexuronic acid as the backbone structure, and the term xe2x80x9csulfated glycosaminoglycanxe2x80x9d means a member of the glycosaminoglycan, which has hexosamine or hexuronic acid having a sulfate group as a constituting monosaccharide, and is a polysaccharide which has at least one hexuronic acid having no sulfate group at the 2-position as a constituting monosaccharide.
Also, the disaccharide composition (a) of the glycosaminoglycan derivative is calculated from values measured by a disaccharide analysis process which will be described later in Test Process 1 of Examples, and the weight average molecular weight (c) is a value measured by a molecular weight measuring process described in Test Process 2 of Examples. Also, the APTT of (b) is a value measured by an APTT measuring process described in Test Process 4 of Examples.
The disaccharide composition defined in (a) shows the ratio of each unsaturated disaccharide having a specified structure, which is calculated by defining the total amount of unsaturated disaccharides represented by the following general formula (7) as 100% (total mol % of 2-acetamido-2-deoxy-4-O-(4-deoxy-a-L-threo-hex-enopyranosyluronic acid)-D-glucose (hereinafter also referred to as xe2x80x9cxcex94DiHS-OSxe2x80x9d), 2-acetamido-2-deoxy-4-O-(4-deoxy-xcex1-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose (hereinafter also referred to as xe2x80x9cxcex94DiHS-6Sxe2x80x9d), 2-deoxy-2-sulfamino-4-O-(4-deoxy-xcex1-L-threo-hex-4-enopyranosyluronic acid) -D-glucose (xcex94DiHS-NS), 2-acetamido-2-deoxy-4-O-(4-deoxy-2-O-sulfo-xcex1-L-threo-hex-4-enopyranosyluronic acid) -D-glucose (hereinafter also referred to as xe2x80x9cxcex94DiHS-USxe2x80x9d), 2-deoxy-2-sulfamino-4-O-(4-deoxy-xcex1-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose (hereinafter also referred to as xe2x80x9cxcex94DiHS-di(6,N)Sxe2x80x9d), 2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-xcex1-L-threo-hex-4-enopyranosyluronic acid)-D-glucose (hereinafter also referred to as xe2x80x9cxcex94DiHS-di(U,N)Sxe2x80x9d), 2-acetamido-2-deoxy-4-O-(4-deoxy-2-O-sulfo-xcex1-L-threo-hex-4-enopyranosyluronic acid)-6-O-sulfo-D-glucose (hereinafter also referred to as xe2x80x9cxcex94DiHS-di(U,6)Sxe2x80x9d) and 2-deoxy-2-sulfamino-4-O-(4-deoxy-2-O-sulfo-xcex1-L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-glucose (xcex94DiHS-tri(U,6,N)S)) that can be identified by the disaccharide analysis described in Test Process 1 in which enzyme digestion and high performance liquid chromatography are used in combination, and the value reflects the position and number of sulfate groups of the glycosaminoglycan derivative before the enzyme digestion.
Also, the structures represented by the above abbreviations may also be shown as follows:
xcex94DiHS-OS: xcex94HexA1xe2x86x924GlcNAc, xcex94DiHS-6S: xcex94HexA1xe2x86x924GlcNAc(6S), xcex94DiHS-NS: xcex94HexA1xe2x86x924GlcNS, xcex94DiHS-US: xcex94HexA(2S) 1xe2x86x924GlcNAc, xcex94DiHS-di(6,N)S: xcex94HexA1xe2x86x924GlcNS(6S), xcex94DiHS-di(U,N)S: xcex94HexA(2S)1xe2x86x924GlcNS, xcex94DiHS-di(U,6)S; xcex94HexA(2S)1xe2x86x924GlcNAc(6S), and xcex94DiHS-tri (U,6,N)S: xcex94HexA(2S)1xe2x86x924GlcNS(6S).
In the above formulae, xcex94HexA means unsaturated hexuronic acid, GlcNAc means N-acetylglucosamine, GlcNS means N-sulfated glucosamine and the parenthesized part shows binding position of sulfate group.
The unsaturated disaccharide having the structure of general formula (7) formed in the disaccharide analysis process is formed from a backbone structure of (A)-(B) in general formula (2) in which the hexuronic acid of general formula (3) or (4) and the hexosamine of general formula (6), which constitute the backbone structure of glycosaminoglycan to be analyzed, are bonded to each other.
The glycosaminoglycan derivatives of the present invention are novel polysaccharides having specified physical properties derived from glycosaminoglycan, but are described herein as glycosaminoglycan derivatives for the sake of convenience.
The glycosaminoglycan derivatives of the present invention are sulfated polysaccharides in which, in the disaccharide composition calculated from the values measured by the disaccharide analysis process described in Test Process 1, mol % of xcex94DiHS-tri(U,6,N)S is from 0 to 10%, preferably from 0 to 5%, and more preferably from 2 to 4%, mol % of xcex94DiHS-di(6,N)S is from 95 to 70%, preferably from 90 to 80%, and more preferably from 82 to 87%, mol % of xcex94DiHS-NS is from 5 to 20%, preferably from 10 to 15%, and more preferably from 11 to 14%, and its weight average molecular weight as a value measured by the molecular weight measuring process described in Test Process 2 is from 9,000 to 13,000 Da, and more preferably from 10,000 to 12,000 Da.
Also, the glycosaminoglycan derivatives of the present invention are glycosaminoglycan derivatives which have a structure formed by the repeating unit of hexosamine and hexuronic acid as the backbone structure. Examples of the hexosamine include glucosamine, galactosamine and mannosamine, and glucosamine is preferred. It is preferred that one or both of the amino group and the 6-position hydroxyl group of hexosamine are sulfated, namely, it is N-sulfated and/or 6-O-sulfated, though there is no hindrance when the hexosamine has no sulfate group. Examples of the hexuronic acid include D-glucuronic acid, L-iduronic acid and the like. It is preferred that a part of hexuronic acid is cleaved between the 2- and 3-position carbon atoms, preferably, the cleaved region is reduced after oxidative cleavage reaction, and that a part or entire portion of the 2-position hydroxyl group of uncleaved hexuronic acid is not substituted with a sulfate group. In addition, one or more of the structural unit represented by general formula (1) in which the cleaved hexuronic acid is bonded to hexosamine in the repeating unit of the backbone structure is present in one molecule of the glycosaminoglycan derivative.
Also, the glycosaminoglycan derivatives of the present invention have a characteristic in that activated partial thromboplastin time (APTT) when measured by adding it to standard blood plasma at a final concentration of 3 xcexcg/ml is 50 seconds or less. The glycosaminoglycan derivatives of the present invention having such a characteristic can be represented by the following general formula (2):
HOxe2x80x94((A)-(B))nxe2x80x94Hxe2x80x83xe2x80x83(2)
In this formula, (A) represents a glucuronic acid residue represented by the following general formula (3), an iduronic acid residue represented by the following general formula (4), or a cleaved hexuronic acid residue represented by the following general formula (5): 
and (B) represents a hexosamine derivative residue represented by the following general formula (6): 
In general formulae (3) to (6), R1 and R3 each independently represents H or SO3H, and R2 independently represents COCH3or SO3H.
Also, a hexosamine residue represented by general formula (6) in which at least one of R1 and R2 is SO3H is present in-molecule of the glycosaminoglycan derivative of the present invention represented by general formula (2).
In addition, in general formula (2), n is an integer which satisfies 15xe2x89xa6nxe2x89xa640, preferably 20xe2x89xa6nxe2x89xa630, and at least one of (A) is a residue of general formula (5).
The glycosaminoglycan derivatives of the present invention (hereinafter referred also to as xe2x80x9cinventive substancesxe2x80x9d) are not particularly limited, so long as they are derivatives of a sugar chain using hexosamine and hexuronic acid as the backbone structure and have a sulfate group, and so long as they satisfy the characteristics of (a) to (d). However, derivatives of heparan sulfate or heparin, particularly derivatives of heparin, are preferred. The inventive substances have a structure in which a part of hexuronic acid is cleaved between the 2- and 3-position carbon atoms. Also, the sulfation ratio of the 2-position hydroxyl group of uncleaved hexuronic acid as a constituent of the inventive substances is less than 10%, preferably less than 5%. The glycosaminoglycan derivatives of the present invention can be represented by general formula (2), wherein n is 15xe2x89xa6nxe2x89xa640, preferably 20xe2x89xa6nxe2x89xa630, namely, they are polysaccharides having from 30 to 80 sugar residues, preferably from 40 to 60 sugar residues. In addition, since the anticoagulation activity of the substances is low, it is preferred that the activated partial thromboplastin time (APTT) when measured by adding them to a standard blood plasma sample collected from a healthy parson at a final concentration of 3 xcexcg/ml is 50 seconds or less.
In the neurite outgrowth promoting activity test described in Examples in which primary culture cells of Wistar rat cerebral cortex cells are used, it is preferred that the glycosaminoglycan derivatives as the inventive substances have 1.5 times or higher activity than the case of standard heparin when culturing is carried out by adding the substances at a final concentration of from 1 xcexcg/ml to 10 xcexcg/ml.
In addition, it is preferred that the inventive substances show markedly stronger sialidase inhibition activity, particularly influenza virus sialidase inhibition activity, than known sialidase inhibitors and standard heparin, when measured by the process described in Examples.
The production process of the present invention is a process for producing glycosaminoglycan derivatives having a repeating unit structure of hexosamine and hexuronic acid as the backbone structure, in which a part of hexuronic acid is cleaved between the 2- and 3-position carbon atoms, preferably, the cleaved region is reduced after cleavage by an oxidative cleavage reaction, and the greater part of the 2-position sulfate group of uncleaved hexuronic acid is desulfated, which comprises the following steps (i) and (ii):
(i) a step in which a glycosaminoglycan having a repeating unit structure of hexosamine and hexuronic acid as the backbone structure is subjected to a cleavage treatment to cleave between only the 2- and 3-position carbon atoms of at least a part of hexuronic acid having no sulfate group on the 2-position, and
(ii) a step in which the product of step (i) is subjected to a desulfation treatment by a desulfation method capable of specifically removing the 2-position sulfate group of hexuronic acid to desulfate 90% or more of sulfate groups of the hexuronic acid having a sulfate group at the 2-position.
1. Sulfated Glycosaminoglycan (Material)
The glycosaminoglycan used in the process of the present invention is a polysaccharide having a repeating unit structure of hexosamine and hexuronic acid as the backbone structure and containing at least one hexuronic acid having no sulfate group at the 2-position as a constituting monosaccharide. Preferred examples of the glycosaminoglycan include heparin, heparan sulfate, and the like, and heparin is particularly preferred, because the sulfation ratio of the 2-position hydroxyl group of hexuronic acid is high and the cleavage of hexuronic acid in the following step (i) can be appropriately controlled.
2. Step (i): Cleavage Treatment of Hexuronic Acid
The xe2x80x9ccleavage treatmentxe2x80x9d described in the step (i) of the process of the present invention is not particularly limited, so long as it is a treatment for specifically cleaving only between the 2- and 3-position carbon atoms of hexuronic acid having no sulfate group at the 2-position among constituting monosaccharides of sulfated glycosaminoglycan. Examples thereof include an oxidation-reduction treatment. This oxidation-reduction treatment is carried out by cleaving between the 2- and 3-position carbon atoms of the specified hexuronic acid through an oxidation reaction using an oxidizing agent to obtain an oxidatively cleaved reaction product and then reducing the aldehyde groups formed in the cleaved region of the hexuronic acid through a reduction reaction treatment. The oxidizing agent is not particularly limited, so long as it is a substance which can achieve the object of this reaction. Examples thereof include a periodate, hydrogen peroxide, and a periodate is particularly preferred. Examples of the periodate include periodic acid alkali metal salts, such as sodium periodate, potassium periodate and the like, and sodium periodate is preferred.
When an oxidation-reduction reaction treatment is carried out as the cleaving treatment, an oxidation reaction is carried out, for example, in a solution containing sodium periodate at a concentration of from 0.01 to 0.3 M preferably from 0.05 to 0.2 M, and the sulfated glycosaminoglycan at a concentration of from 0.5 to 10%, preferably from 1 to 7%, at a pH of from 3 to 7, preferably from 4 to 5, and at a treating temperature of from 0 to 37xc2x0 C., preferably from 0 to 10xc2x0 C., more preferably 4xc2x0 C., for at least 1 day, preferably 3 days. After the oxidation reaction, excess sodium periodate is decomposed through a reducing reaction by adding from 100 to 500 mM ethylene glycol or glycerol. Thereafter, an oxidative cleavage reaction product of hexuronic acid can be obtained by carrying out dialysis or further employing freeze-drying or ethanol precipitation process as occasion demands.
Thereafter, the oxidation-reduction reaction treatment of hexuronic acid is completed by further reducing the aldehyde groups formed in the cleaved region of hexuronic acid by periodic acid oxidation. The reduction of aldehyde groups can be carried out using a reducing agent such as an alkali metal borohydride or lithium aluminum hydride, and the reducing agent is preferably an alkali metal borohydride, most preferably sodium borohydride. When sodium borohydride is used as the reducing agent, it is preferred to carry out the reaction, for example, in a solution of pH 8.5 to 9.5 containing from 0.1 to 0.5 M, preferably 0.2 M, of sodium borohydride and from 1 to 20%, preferably from 5 to 10% of the oxidative cleavage reaction product (w/v), at 4xc2x0 C. for 3 hours. It is preferred to obtain a sodium salt of the periodic acid oxidation-reduction reaction product by further decomposing excess sodium borohydride by adjusting the reaction solution to pH 4 to 5 and then terminating the reduction reaction by dialyzing it against distilled water.
Particularly, when heparin is used as the material, it is preferred to carry out the cleavage treatment by an oxidation-reduction reaction treatment using a periodate and an alkali metal borohydride, and a periodic acid oxidation-reduction heparin is obtained by this treatment as a periodic acid oxidation reduction product of heparin.
The hexuronic acid cleavage product of sulfated glycosaminoglycan obtained by the treatment to specifically cleave hexuronic acid among the constituting monosaccharides of sulfated glycosaminoglycan is subjected to the subsequent step (ii) in which the 2-position sulfate group of at least a part of hexuronic acid is desulfated.
3. Step (ii): Desulfation Treatment
The xe2x80x9cdesulfation treatmentxe2x80x9d described in the step (ii) of the process of the present invention can be carried out without particular limitation, so long as it is a treatment (selective desulfation treatment) to selectively desulfate the 2-position sulfate group of the hexuronic acid having a sulfate group at the 2-position, which constitutes the xe2x80x9chexuronic acid cleavage product of sulfated glycosaminoglycanxe2x80x9d formed in the step (i). When complete desulfation is desired, it is preferred to employ a process which is carried out under alkaline conditions, namely a process by hydrolysis reaction, particularly a process which includes a step in which the hexuronic acid cleavage product of sulfated glycosaminoglycan obtained in the step (i) is dissolved in an aqueous solution of an alkali metal hydroxide, such as sodium hydroxide, potassium hydroxide, or the like, or an aqueous solution of an alkaline earth metal hydroxide, such as magnesium hydroxide, calcium hydroxide, or the like, preferably an aqueous alkali metal hydroxide solution, most preferably an aqueous sodium hydroxide solution, and the resulting solution is immediately frozen to carry out freeze-drying.
Specifically, a solution of the hexuronic acid cleavage product of glycosaminoglycan is prepared by dissolving the hexuronic acid cleavage product of glycosaminoglycan in an aqueous alkali metal hydroxide solution or aqueous alkaline earth metal solution having a concentration of from 0.0125 to 0.2 N, preferably at ice-cooling (0xc2x0 C.) to room temperature (24xc2x0 C.), and then the resulting solution is freeze-dried. Thereafter, it is preferred that the freeze-dried product is again dissolved in an aqueous alkali metal hydroxide solution or aqueous alkaline earth metal solution (0.0125 to 0.2 N) to a concentration of from 1 to 2.5 M, preferably 2 M, and the resulting solution is adjusted to pH 8 to 10 with an acid, preferably a weak acid, more preferably a carboxylic acid, such as acetic acid or the like, and immediately subjected to dialysis against distilled water for at least 1 day, preferably 2 days, followed by freeze-drying or ethanol precipitation.
Desulfation of the hexuronic acid cleavage product of sulfated glycosaminoglycan is carried out in such a manner that generally 70% or more, preferably 90% or more, more preferably 95% or more, of the 2-position sulfate groups of uncleaved hexuronic acid are removed.
According to the process of the present invention, when the partial desulfation treatment is carried out using the periodic acid oxidation-reduction heparin obtained in the step (i) from heparin as the material sulfated glycosaminoglycan, the partially 2-O-desulfated periodic acid oxidation-reduction heparin of the present invention is obtained.
A schematic illustration of the reaction steps for the production of the glycosaminoglycan derivative of the present invention from heparin as the starting material by the cleavage treatment of step (i) and the desulfation treatment of step (ii) is shown in FIG. 1. In the drawing, (a) is a schematic representation of heparin, (b) is a reaction product in which aldehyde groups are formed by the oxidative cleavage reaction of hexuronic acid, (c) is a reduction reaction treated product obtained by the reduction reaction treatment of the aldehyde groups of (b), and (d) is a glycosaminoglycan derivative of the present invention in which the 2-O-sulfate group is selectively desulfated.
Composition 1 of the present invention is a therapeutic agent for neurological disorders, comprising, as an active ingredient, the inventive substance, a glycosaminoglycan derivative or a pharmaceutically acceptable salt thereof.
The glycosaminoglycan derivatives having neurite outgrowth promoting activity are useful as a pharmaceutical composition using the activity, such as a neuronal disease treating agent for, for example, central nerve diseases (e.g., Alzheimer disease, ischemic dementia, etc.) or peripheral nerve diseases (e.g., diabetic neuropathy, alcoholic neuropathy, amyotrophic lateral sclerosis (ALS), peripheral nerve damage caused by injury or side effect of medicament, Guillain-Barre syndrome, etc.).
Also, the glycosaminoglycan derivatives of the present invention which are the active ingredient of Composition 1 of the present invention can also be applied to therapeutic and preventing agents as a nervous function reduction inhibitor, because they can prevent reduction of nervous functions accompanied by nerve degeneration, by inhibiting the activity of sialidase to degrade cell membrane ganglioside which is concerned in the maintenance of neural cell survival, synaptic function and axon function.
Composition 2 of the present invention is a sialidase inhibitor, comprising, as an active ingredient, the inventive substance, a glycosaminoglycan derivative or a pharmaceutically acceptable salt thereof.
The glycosaminoglycan derivatives having sialidase inhibition activity as the inventive substances can be used as a sialidase inhibitor using its activity. Particularly, since they can inhibit growth of viruses by inhibiting viral sialidase, they can be used as an anti-viral agent, particularly as an anti-influenza remedy. Particularly, the inventive substances show markedly more excellent sialidase inhibition activity than known sialic acid derivatives used as influenza remedies.
The term xe2x80x9cremedyxe2x80x9d as used herein includes not only drugs for improving conditions of patients into normal states and drugs for alleviating diseases but also a concept of xe2x80x9cdrugs for preventionxe2x80x9d for preventing infection with and contraction of diseases.
Examples of the pharmaceutically acceptable salts of the glycosaminoglycan derivatives, which are used as the active ingredient of Compositions 1 and 2 of the present invention include pharmaceutically acceptable salts among amine salts, quaternary ammonium salts, alkali metal salts and alkaline earth metal salts. Specific examples include sodium salts, potassium salts, calcium salts, magnesium salts, and the like, and sodium salts and potassium salts as alkali metal salts are preferred, and sodium salts are more preferred, for example, in view of the affinity for the living body.
The glycosaminoglycan derivatives of the present invention show a characteristic of being lower in anticoagulation activity than standard heparin. In especially desirable glycosaminoglycan derivatives, it is most preferred that the APTT value becomes 50 seconds or less when measured in accordance with the process shown in Test Example 4 which will be described later, by setting the concentration in the measuring solution (final concentration) to 3 xcexcg/ml. Also, regarding the glycosaminoglycan derivative (the inventive substance) used in the composition of the present invention as a pharmaceutical composition, it is preferred that at least one of thrombin time (hereinafter referred to as xe2x80x9cTTxe2x80x9d) calculated by a TT measuring process which will be described later and APTT activity calculated by the APTT measuring process becomes 5% or less in comparison with standard heparin. Still more preferably useful is a substance having a relatively low TT activity, in which the TT activity and the APTT activity are both 3% or less in comparison with standard heparin and both of these activities have a relationship of 0xe2x89xa6TT activity/APTT activityxe2x89xa60.5, because low bleeding activity can be expected when used in a pharmaceutical composition.
The term xe2x80x9cstandard heparinxe2x80x9d as used herein means a substance identical to the standard heparin described in Examples.
When a pharmaceutical composition containing the inventive substance as the active ingredient is administered to the living body, the dosage form and route of administration can be optionally selected in response to the properties and seriousness of the disease to be treated. For example, it can be safely administered parenterally or orally to a warm-blooded animal (e.g., human, mouse, rat, hamster, rabbit, dog, cat, horse, etc.) as it is or as pharmaceutical composition preparations together with other pharmaceutically acceptable additives, such as carriers, fillers, diluents and the like (e.g., injections, suppositories, tablets, capsules, solutions, ointments, gels, etc.).
Regarding the administration form of the therapeutic agent for neuronal diseases, parenteral administration is preferred, injections can be cited as the dosage form suitable for the administration form, and intravenous injection or drip infusion can be exemplified as preferred mode of the administration process, though not limited thereto.
Regarding the administration form of the influenza remedy, any of its oral and parenteral administration forms can be optionally selected. Examples of the dosage form suitable for oral administration include powders, granules, tablets, capsules, aerosols and sprays, and solutions can be exemplified as the dosage form suitable for parenteral administration. In addition, as the dosage form of the influenza remedy particularly for prevention, solutions used by oral administration are also preferred, and administration of the solutions using a spray or the like can be cited as a preferred process, though not limited thereto.
A formulating amount and a dose of the glycosaminoglycan derivative as the active ingredient of the preparation are not particularly limited, because they should be decided individually depending on, for example, the administration process, administration form and using purpose of the preparation and illustrative symptoms and body weight of each patient, but in the case of intravenous injection, approximately from 100 xcexcg/kg to 100 mg/kg per day can be exemplified as the amount of the glycosaminoglycan derivatives to be administered to a patient. Also, regarding the administration frequency of the preparation, once a day is possible, and it can also be administered by dividing the daily dose into 2 to 4 or more doses per day. It is also possible to administer it continuously by, for example, drip infusion.
In this connection, the glycosaminoglycan derivative as the active ingredient of the pharmaceutical composition showed no toxicity upon cells in the following Examples. It is known that the LD50 value of heparin by an acute toxicity test in mice (males and females) is 5,000 mg/kg or more by oral administration, 2,500 mg/kg or more by subcutaneous or intraperitoneal administration and about 1,000 mg/kg by intravenous injection. Since the glycosaminoglycan derivatives as the active ingredient of the pharmaceutical composition of the present invention show markedly low APTT activity and TT activity of both less than 3% in comparison with the standard heparin, this fact also supports high safety of the glycosaminoglycan derivatives (the inventive substances) which are the active ingredient of the pharmaceutical composition.